Ezine

Published: Sep 15, 2011

Author: Steve Down

Channels: Base Peak

Under compromising conditions

Invasive aspergillosis is a disease caused by spores of the pathogen Aspergillus fumigatus, which are present in normal everyday air. Most healthy people are naturally resistant to infection but it is a different story for the immunocompromised.

Patients undergoing chemotherapy or radiotherapy, suffering from AIDS, or taking immunosuppressive drugs following organ transplant are far more likely to become infected, with life threatening consequences. The disease progresses rapidly and has a mortality rate of up to 90%.

The most common form of the disease is pulmonary aspergillosis but other tissues apart from the lungs, such as the central nervous system, sinuses, heart and kidney, can be affected.

As with most diseases, early detection gives a better chance of successful treatment, in this case using antifungal drugs. However, current diagnoses for invasive aspergillosis leave a lot to be desired, according to a team of scientists from New Zealand and Germany.

Amy Scott-Thomas, Shrawan Bhandari and John Pearson from the Department of Pathology at the University of Otago, Stephen Chambers from the Canterbury District Health Board in Christchurch and Mona Syhre from the Department for Inner Medicine, Neurology and Dermatology at Leipzig University claimed that methods such as biopsy, bronchoscopy and PCR of blood samples "have significant limitations."

They have been investigating an alternative approach for the diagnosis of pulmonary invasive aspergillosis based on earlier research by Syhre and colleagues who discovered that infected immunocompromised patients emit 2-pentylfuran (2PF) on their breath. Unfortunately, the sensitivity and specificity were just 77 and 78%, respectively, suggesting that false positives might be at play.

In new work, Scott-Thomas and coworkers have looked for external sources of 2PF which might influence its appearance and affect the test, as well as other mitigating factors.

Interfering foodstuffs

It was already known that 2PF is present in certain foods, either naturally or as an added flavour enhancer to impart a green bean, metallic or vegetable note. So, the team examined many different types of foods and beverages to see if they might interfere with 2PF breath testing. In addition, ten cosmetics products and four antifungal medications were studied.

2PF is a volatile compound, so it was collected from the samples by SPME for subsequent analysis by GC/MS. A polar polyethylene glycol column was used to separate 2PF from the other volatiles present and it was detected by electron ionisation in an ion trap mass spectrometer.

The presence of 2PF was confirmed from its MS/MS spectrum and it was quantified using the signal responses for ions at m/z 53 and 81. The quantification limit was 1.13 attograms and the calibration curves for both ions displayed good linearity.

Of the 45 foods tested, ten contained 2PF. They included three brands of soy milk and a soy-based nutritional supplement often given to hospitalised patients. Peanuts, tinned asparagus, rolled oats, pumpkin and a commercial yeast extract (MarmiteTM) also tested positive. 2PF was absent from coffee beans or parsley leaves despite previous reports of its presence.

No 2PF was found in the cosmetic products. Mouthwashes, toothpaste, aftershave, perfume, shampoos, soap products and vaseline all tested blank. The medications fluconazole, itraconazole, amphotericin B and voriconazole, in powdered or liquid form, also tested negative.

The data clearly indicate that the diet of the patient should be taken into account when breath testing for 2PF as an indicator of invasive aspergillosis. The food-based concentrations were relatively small but could still influence the results of a test to give a false positive.

Fasting effects

In a further step, the researchers conducted tests on healthy subjects by asking them to exhale into collection bags. They each provided two samples, the first following 12 hours of fasting and the second taken the next day after their normal food regime, but avoiding the foods known to contain 2PF. In addition, some subjects gave breath samples twice on the same day.

It was clear from the results that there is no diurnal effect in healthy volunteers and that fasting for 12 hours did not affect the method specificity for 2PF. So, no overnight fasting is required.

Experiments with soy milk revealed that rinsing the mouth with water at least 30 minutes after ingestion was sufficient to remove any food-based traces.

In the long term, more foods should be tested to rule out potential interferences. Meanwhile, the team concluded that the GC/MS method for measuring 2PF in the breath of patients with invasive aspergillosis is satisfactory down to attogram levels, provided that they keep a food diary to ensure the absence of 2PF-containing foods in the diet.

The views represented in this article are solely those of the author and do not necessarily represent those of John Wiley and Sons, Ltd.

Soymilk is one of the foodstuffs that interfere with the breath test for 2-pentylfuran